MS coupled with LC is a common approach to quantifying compounds in a sample. The quantitation is typically performed by first separating compounds by LC to generate a sequence of chromatograms, each associated with a retention time (RT) window; next, ionizing and detecting the separated compounds by MS to produce a plurality of mass spectra having ion peaks; then, using a peak area or a sum area of all peaks within a mass window, associated with a target compound, to infer quantitative information about the compound, assuming there is a correlation between the peak area and the compound concentration.
This approach, however, faces difficult challenges in quantifying a target compound in a complex sample, for it is based conceptually upon two assumptions: first, all ion peaks used in quantitation are associated with a target compound; second, all ion peaks used in quantitation are shaped and positioned as theoretically predicted. However, these assumptions do not hold in quantifying compounds intertwined in a complex sample, because as the complexity (or/and dynamic range) of a sample increases, the chance of multiple compounds co-eluting from LC in a same RT window is also magnified, resulting in mass spectra, where ions from different compounds occupy a same mass-to-charge-ratio (m/z) space and interfere with each other. Moreover, as the complexity of a sample increases, the number of high-abundance compounds in the sample proportionally increases as well, which can saturate a detector and corrupt linearity of detector response, given that there will always exist an instrumental limit of detection. Accordingly, a signal profile is no longer correlated with the quantity of the compounds, as their concentration increases beyond a level that saturates a detector.
The term “complex sample,” as used herein, refers to a sample that contains a multitude of naturally occurring or man-made biological components, such as proteins, peptides, metabolites, lipids, antibodies in serum, or mixtures thereof.